The peak wavelengths of the absorption and emission spectra of the nanocrystal dispersion were blue and red shifted, respectively, compared with those of tetrahydrofuran solution. Powder X-ray diffraction analysis confirmed the crystallinity of the nanocrystals. The presented 1,4-bis(4-methylstyryl)benzene nanocrystals are expected to be a candidate for a new class of optoelectronic material.Single-crystal 1,4-bis(4-methylstyryl)benzene is a promising material for optoelectronic device applications. We demonstrate the preparation of 1,4-bis(4-methylstyryl)benzene nanocrystals by a wet process using a bottom-up reprecipitation technique. Scanning electron microscopy revealed the morphology of the nanocrystals to be sphere-like with an average particle size of about 60 nm. An aqueous dispersion of the nanocrystals was monodisperse and stable with a  Recently, organic single crystals have attracted considerable attention for optoelectronic device applications because of their high stimulated cross-sections, broad and high-speed nonlinear optical responses, and broad tuning wavelength . In addiIn contrast, we have investigated the preparation and evaluated the properties of nano-sized organic crystals, i.e., organic nanocrystals -11. OrgaThe aim of this study is to demonstrate the preparation of BSB-Me nanocrystals having narrow size distribution with singular morphology by means of a bottom-up, wet process using the reprecipitation method. This method makes it possible to control the particle size and morphology of the nanocrystals. We prepared BSB-Me nanocrystal dispersions in water, and investigated the size, morphology, optical properties, and powder X-ray diffraction pattern of the nanocrystals.BSB-Me (>98.0%) was purchased from Tokyo Chemical Industry Co., Ltd.  and used without further purification. Tetrahydrofuran (THF) (>99.5%) was purchased from Wako Pure Chemical Industries, Ltd. . Purified water (18.2 MΩ) was obtained from a Milli-Q A-10 .BSB-Me was dissolved in THF (2 mM) at 50°C, and 100 μl of the solution was injected into vigorously stirred  poor solvent water (10 ml at 24°C) using a microsyringe. As a result, the BSB-Me suddenly precipitated to form dispersed nanocrystals. Syringe filter  was used to remove small degree of aggregates from the nanocrystal dispersion.ζ-potential of the nanocrystal dispersion were evaluated using an ELSZ-1000 zeta-potential and particle size analyzer . Ultraviolet-visible (UV-vis) absorption spectra and fluorescence spectra were measured using a V-550 UV/vis spectrophotometer  and F-2500 fluorescence spectrophotometer , respectively.The particle size and morphology of the BSB-Me nanocrystals were evaluated using scanning electron microscopy . To prepare specimens for imaging, the nanocrystals were collected from the water dispersion using suction filtration with a membrane filter (0.05-μm pore size), followed by platinum sputter coating . The average particle size, size distribution, and n = 211)  compared with that of the THF solution (λmax = 359 nm) , leading to a blue shift compared with in solution [λmax = 466 nm) compared with that of the solution state (λmax = 415 nm)  and the THF solution (λmax = 359 nm) almost corresponded to those of the respective absorption spectra  are matched at the excitation wavelength. The quantum yield of the unknown is calculated using Equation 1:To investigate the photoluminescence efficiency of the BSB-Me nanocrystal water dispersion, we estimated its photoluminescence quantum yield. The manner to estimate the quantum yield of a fluorophore is by comparison with standards of known quantum yield. We used the standard of BSB-Me dichloromethane solution referred in the literature , in whicQ is the quantum yield, I is the integrated intensity (areas) of spectra, OD is the optical density, and n is the refractive index. The subscripted R refers to the reference fluorophore of known quantum yield. The data of I and OD were obtained from Figure where θ = 9.0 and 13.6, corresponding with those previously reported for single bulk crystals [θ = 20.9, 23.6, 28.4, and 29.4 which did not correspond with any previously observed peaks for single crystals [θ = 9.0 and (006) in 2θ = 13.6, the nanocrystals basically had planar structure, supporting the occurrence of H-aggregation according to the work of Kabe et al. [θ = 20.9, 23.6, 28.4, and 29.4, the nanocrystals may have had slightly different crystal structure than the bulk single crystal. Actually, we have previously reported the existence of a softened crystal lattice in nanocrystals [The crystallinity of the BSB-Me nanocrystals was confirmed using powder X-ray diffraction analysis Figure . Two strcrystals ,35. A sicrystals ,36. Thatζ-potential of -41.62 mV using ELSZ-1000. The blue shift and red shift of maximum peak wavelength were observed in absorption and emission spectra, respectively. This optical feature may have arisen from supramolecular interactions like those caused by the herringbone structure, i.e., H-aggregation, in the nanocrystals. The photoluminescence quantum yield of the BSB-Me nanocrystal water dispersion was estimated to be 9.2 ± 0.1%. Powder X-ray diffraction analysis confirmed the crystallinity of the BSB-Me nanocrystals. In future work, these BSB-Me nanocrystals will be applied to crystalline-based optoelectronic devices. Measuring amplified spontaneous emission and nonlinear optical properties of single nanocrystals will be a particularly interesting topic for the near future. We will also investigate and discuss elsewhere the nanocrystal size distribution using Scherrer's equation based on the data of XRD measurements. Further detailed optical properties such as an absolute photoluminescence quantum yield, fluorescence lifetime, and radiative decay rates of BSB-Me nanocrystals will be discussed elsewhere. Furthermore, fluorescent BSB-Me nanocrystals could be used in biological applications such as fluorescent bioimaging of cells and tissue similar to that in our previous work.We demonstrated the preparation of a BSB-Me nanocrystal dispersion in water by the reprecipitation method, which is a bottom-up, wet process for preparing organic nanocrystals. SEM observations revealed that the nanocrystals had a sphere-like morphology. The average particle size was 60.9 nm, measured using an ELSZ-1000 zeta-potential and particle size analyzer. The nanocrystal dispersion was stable with a measured The authors declare that they have no competing interests.KB contributed to the conception of the study, carried out all the experiments, and drafted the manuscript. KN contributed to the interpretation of the data and revision of the manuscript. Both authors read and approved the final manuscript.KB is an Endowed Chair Associate Professor at the Department of Visual Regenerative Medicine, Osaka University Graduate School of Medicine, Japan, and KN is a Professor and a medical doctor at the Department of Ophthalmology, Osaka University Graduate School of Medicine, Japan.


Early and rapid detection of dengue virus (DENV) infection during the febrile period is crucial for proper patient management and prevention of disease spread. An easy to perform and highly sensitive method is needed for routine implementation especially in the resource-limited rural healthcare settings where dengue is endemic.A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes. The sensitivity and specificity of the RT-LAMP were evaluated. The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. The test results of RT-LAMP were statistically compared to those of quantitative reverse transcription-polymerase chain reaction (qRT-PCR), IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA).Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses.The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods. Dengue virus (DENV) is a positive-sense single-stranded RNA virus with a genome of ~10.7 kb in length  and eachRoutine laboratory diagnosis of DENV infection in most resource-limited countries is based on the detection of virus-specific antibodies as well as virus isolation. Detection of viral components such as non-structural protein 1 (NS1) and virus genomic RNA are done but mostly in referral or in more well-funded diagnostic laboratories -18. AlthThe loop-mediated isothermal amplification (LAMP) of genomic sequence is a novel method for the detection of nucleic acid with high specificity and sensitivity without the need of specialized equipment -35. The Aedes albopictus) mosquito cells for one week and virus RNA was extracted from the infected cell culture supernatant.A total of 11 reference DENV strains  were used in this study ,39-41. AThe study obtained ethics approval from the UMMC Medical Ethics Committee (Ethics Committee/IRB Reference Number: 908.11). The study was conducted during the period from October to December 2012. A total of 305 serum samples from patients clinically suspected with DENV infection were obtained. All clinical samples were taken as part of standard patient care. The samples were divided in the laboratory to be used for routine diagnosis by serological assays and evaluation of the RT-LAMP assay developed in this study.Total RNA was extracted from 140 μl of infected culture supernatant or patient serum samples using QIAamp Viral RNA Mini Kit , following the manufacturer’s protocol. All RNA extraction was done manually. The RNA was eluted in 60 μl of nuclease-free water and stored at −80°C until needed.et al.[The DENV-specific primers used for RT-LAMP assay were designed from the 3′UTR of the DENV genome. The 3′UTR nucleotide sequences of each DENV serotypes were retrieved from GenBank. Multiple sequence alignment of the 3′UTR sequences was performed using Clustal X 2.0 . A set oet al.  added with 20 pmol each of inner primers FIP/123, FIP/4, BIP/123, and BIP/4; 2.5 pmol each of outer primers F3/134, F3/2, B3/123, and B3/4; 20 pmol of loop primer BLP/1234; 1 μl of Fluorescent Detection Reagent ; and 5 μl of the extracted RNA template. A positive control using 1000 copies (determined by qRT-PCR) of DENV RNA extracted from culture supernatant and a negative control (nuclease-free water) were included in each run. The RT-LAMP reactions were incubated at 63°C for 80 min and inactivated at 80°C for 5 min in LA-500 Loopamp real-time turbidimeter . The turbidity of RT-LAMP reaction was spectrophotometrically recorded at 650 nm every 6 s. The threshold time (Tt) value for positivity by RT-LAMP was determined when the turbidity increased above the threshold value, which was fixed at 0.07 absorbance units. The RT-LAMP amplification was also visually monitored for color change. Positive reaction turned the reaction mix green and fluoresces under the white light and UV irradiation, respectively. The reaction mix remained orange and non-fluorescent in the absence of amplification.The specificity of the RT-LAMP amplification was assessed by single site restriction enzyme digestion of the amplified DNA fragments using BanII . Followigenesig Real-Time qRT-PCR DENV Detection Kit . The qRT-PCR assay standard plot, ranged from 10 to 106 RNA copies, was made by preparing a 10-fold serial dilution of the genesig DENV RNA standard. The genesig DENV RNA standard was a synthetic RNA template with known copy number. The qRT-PCR was performed in a final volume of 20 μl containing 10 μl of real time master mix, 1 μl of probe/primer mix, 4 μl of nuclease-free water, and 5 μl of diluted RNA. Quantitative PCR measurement was performed using StepOnePlus real time PCR system  according to the following condition: 10 min at 55°C, 8 min at 95°C followed by 50 cycles of amplification . Raw data was analyzed with StepOne Software v2.2.1 to determine copy number based on the threshold cycles (Ct). The efficiency of the qRT-PCR was measured from the slope of standard curve.The sensitivity of RT-LAMP assay was assessed using a panel of serially diluted viral RNA  extracted from culture supernatant. The sensitivity test of RT-LAMP was repeated twelve times. The viral RNA used was quantitated using the The clinical applicability of a single-tube RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 serum samples freshly obtained from dengue-suspected patients. The qRT-PCR, as described above, was used as a reference assay for the detection of DENV RNA in the samples. In addition, the samples were simultaneously screened for the presence of anti-dengue IgM using the SD Dengue IgM Capture ELISA kit . The IgM-negative samples were further screened for the presence of anti-dengue IgG using the SD Dengue IgG Capture ELISA kit . The test results of RT-LAMP, qRT-PCR, and ELISA were compared. Acute DENV infection was confirmed by the positive detection of anti-DENV IgM by ELISA and/or the presence of DENV RNA detected by qRT-PCR assay. Serum sample tested positive only for anti-DENV IgG was identified as indicative of past DENV infection.In this study, the results obtained by molecular diagnosis of dengue were not used for the standard patient care as the molecular assay was still being developed. The qRT-PCR was used only as research tool in the reference laboratory. Currently, only the serological methods were approved for routine clinical diagnosis of dengue at the UMMC.http://ktclearinghouse.ca/cebm/toolbox/statscalc).All statistical analysis was performed using IBM SPSS Statistics, version 21 . The degree of agreement between RT-LAMP and qRT-PCR test results was measured by kappa value (κ). Chi-square test  was performed to evaluate and compare the sensitivity of all molecular and serological methods used. In the present study, the p-value <0.001 was used to suggest significant results. The diagnostic performance of RT-LAMP assay as compared to qRT-PCR was calculated using web-based CEBM Statistics Calculator , four inner , and one loop primers (BLP/1234) were designed. This was done using the alignment of the conserved 3′UTR regions of DENV genome  by either qRT-PCR or dengue IgM ELISA, or both  developed a four-tube RT-LAMP assay which employed serotype-specific primers targeting the proximal half of the 3′UTR of DENV genome [et al. developed a single-tube reaction system for the detection of DENV infection using RT-LAMP primers derived from the C-prM gene [The RT-LAMP assay developed here differs from those reported earlier in at least two major areas -38. PariV genome . The fouprM gene ,39-41. IThe RT-LAMP assay developed in this study has a detection limit of at least 100 copies of viral RNA Figure . We defiHere, the RT-LAMP assay for DENV detection was prospectively evaluated for the first time with a large number of clinical serum samples (n = 305) from dengue-suspected patients. The performance of the RT-LAMP assay was validated by simultaneous testing of the samples using real time qRT-PCR, which is known as the most sensitive and specific method for the detection of viral RNA . In our Currently, in most dengue endemic region where resources are limited, early diagnosis of dengue relies very much on the clinical presentations. A number of other infections, however, can present with almost similar features especially during the early febrile phase. Rapid confirmation of dengue is important to ensure proper management of patients and appropriate outbreak prevention measures. Currently, serological assay is the most common method used to confirm DENV infection. In our study, using actual clinical samples when febrile dengue patients first visit to the hospital, 46.8% (80 of 171) of the specimen received indicated that the patients were viremic with the absence of IgM or IgG Figure . The RT-The RT-LAMP assay developed in our study showed high sensitivity and specificity comparable to qRT-PCR for detection of DENV in clinical samples. The single-tube RT-LAMP assay utilized a primer set with coverage for all the reported DENV strains particularly those common in the region. Implementation of the RT-LAMP assay into routine dengue diagnosis to complement the antibody detection would greatly enhance the diagnostic coverage of suspected dengue cases without the need for costly equipment and reagents. The RT-LAMP assay developed in this study is sensitive, specific and simple to perform, and in combination with the ELISA is a promising detection tool for early dengue diagnosis in rural clinics and field situation where resources are limited.All authors have no competing interests.BTT, SSS, KKT, JJ, MBD, and SAB conceived the study and participated in its design and coordination. BTT designed the RT-LAMP primers. BTT and SSS performed sample preparation and RNA extraction. BTT, SSS, KKT, PSH, and RME performed the RT-LAMP, qRT-PCR and ELISA assays. BTT, SSS, KKT, and PSH analyzed the results. SSS carried out the statistical analysis. BTT, SSS and SAB drafted the manuscript. SAB is the principle investigator of this study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/387/prepubMap of RT-LAMP primers in alignment with the DENV 3’UTR consensus sequences. The arrows indicate the orientation of primers in 5′ to 3′ direction.Click here for file


Anopheles funestus s.s and to Anopheles gambiae s.l. (mainly Anopheles gambiae s.s. and Anopheles arabiensis). Several studies have demonstrated that the immune response against salivary antigens could be used to evaluate individual exposure to mosquito bites. The aim of this study was to assess the use of secreted salivary proteins as specific biomarkers of exposure to An. gambiae and/or An. funestus bites.Malaria transmission occurs during the blood feeding of infected anopheline mosquitoes concomitant with a saliva injection into the vertebrate host. In sub-Saharan Africa, most malaria transmission is due to An. gambiae (gSG6 and g-5′nuc) and An. funestus (fSG6 and f-5′nuc) were selected and produced in recombinant form. The specificity of the IgG response against these salivary proteins was tested using an ELISA with sera from individuals living in three Senegalese villages  that had been exposed to distinct densities and proportions of the Anopheles species. Individuals who had not been exposed to these tropical mosquitoes were used as controls .For this purpose, salivary gland proteins 6 (SG6) and 5′nucleotidases (5′nuc) from Anopheles species and against g-5′nucleotidase from An. gambiae, were significantly higher in Senegalese individuals compared with controls who were not exposed to specific Anopheles species. Conversely, an association was observed between the level of An. funestus exposure and the serological immune response levels against the f-5′nucleotidase protein.The IgG responses against SG6 recombinant proteins from these two Anopheles salivary antigenic protein that could be considered to be a promising antigenic marker to distinguish malaria vector exposure at the species level. The epidemiological interest of such species-specific antigenic markers is discussed.This study revealed an  Anopheles densities, these tools lack important logistics and present limited efficiency in the context of low-level exposure to Anopheles bites. In addition, they are not designed for the assessment of the heterogeneity of mosquito exposure at the individual level, +0.65 [0.53 to 0.76] and +0.58 [0.49 to 0.66] for Diama, Dielmo and NDiop, respectively) compared with un-exposed control individuals . Conversely, despite distinct exposure levels to Anopheles bites in these three Senegalese villages, significant differences were detected only between Diama and Dielmo . Although the highest exposure to Anopheles bites was in Dielmo, followed by NDiop and finally Diama; the seroprevalence (a seropositivity cut-off was set as the mean aOD of un-exposed controls + 3 SDs: 0.164 + (3 x 0.130) = 0.554), with values of 55%, 52% and 37% in Dielmo, NDiop and Diama, respectively, was not found to be significantly different among the three villages , NDiop  and Diama  compared with un-exposed control individuals . As observed for gSG6, a comparison of the IgG responses between two Senegalese villages, indicated a significant difference only between Diama and Dielmo . The seroprevalence from Diama (17%), Dielmo (11%) and NDiop (22%) (the seropositivity cut-off was set as the mean aOD of un-exposed controls + 3 SDs: 0.148 + (3 × 0.146) = 0.586) was not found to be significantly different among the three villages  was observed between the IgG responses against g-SG6 and f-SG6 among the exposed individuals. Despite the variations of Anopheles species proportion among the three villages, the large majority of individuals were “equivalently” distributed around the best-fit line. Thus, these observations strongly support a wide cross-reactivity to the gSG6 and fSG6 salivary antigens, which is in agreement with a previous study were +0.50 [0.40 to 0.61], +0.51 [0.39 to 0.63] and +0.65 [0.52 to 0.77] for Diama, Dielmo and NDiop, respectively) compared with un-exposed control individuals . Conversely, no significant differences in the anti-g-5′nuc IgG responses was found, regardless of which two groups from these three Senegalese villages were being compared . Although a higher seroprevalence (the seropositivity cut-off was set as the mean aOD of un-exposed controls + 3 SDs: 0.163 + (3 x 0.147) = 0.604) was detected among individuals living in the NDiop area (40%), where An. gambiae s.l. is largely predominant, compared with Diama (28%) and Dielmo (29%), no significant differences were found among the three villages  and NDiop , compared with un-exposed control individuals  or with individuals living in Diama . No significant difference was noted between individuals living in Diama  and un-exposed control individuals . The seroprevalence (the seropositivity cut-off was set as the mean aOD of un-exposed controls + 3 SDs: 0.219 + (3 x 0.210) = 0.849) was found to be significantly different among individuals living in Dielmo (47%) and those living in Diama (17%)  was not significantly different when compared with the two other Senegalese villages .For the f-5′nuc protein, the IgG responses were significantly different among the four groups  and Dielmo , respectively . A high dispersion of the dots was visible on the scatter plot  could possibly possess antigenic sequences that are absent in f-5′nuc and could explain the differences in serological recognition. Moreover, the g-5′nuc amino-terminal sequence possesses several cysteines (n = 7) that may be involved in protein folding, which could change the epitope conformation or mask some epitopes in the carboxy-terminal . Indeed, the resulting cryptic epitopes in the g-5′nuc protein could be, in contrast, accessible to an antibody response in the f-5′nuc protein and, therefore, induce a “species”-specific serological response.To assess the cross-reactivity level of the IgG response of exposed individuals from the three Senegalese villages (n = 134) against 5′nuc orthologs, a Spearman’s rank correlation coefficient (rho) test was used, and the corresponding Anopheles species behavior is of major importance in vector control interventions to protect human populations against malaria. Anopheles gambiae s.l. and An. funestus share particularly anthropophilic tendencies that contribute to their vectorial capacity[An. gambiae s.l. declined in some Sub-Saharan Africa areas, whereas the An. funestus abundance remained maximal, extending the period of malaria transmission. Therefore, the determination of human exposure to malaria vectors at the quantitative level  and at the qualitative level  should help to adapt malaria control strategies according to the spatial and temporal density of mosquito fauna[Understanding the complexity of the capacity. Howevercapacity. Indeed,to fauna.Anopheles species and to distinguish vector exposure at the species level[To this end, the analysis of the human antibody response against mosquito salivary antigens has proved to be a relevant tool to assess host/vector contact,17,59. ATherefore, the production of specific mosquito saliva antigens in a recombinant form or by using synthetic peptides is a promising alternative strategy for producing safe and highly standardized antigens on a large scale,24. A gaAnopheles Cellia subgenus made the SG6 proteins the first anopheline salivary candidates tested for the exploration of the relationship between levels of anti-gSG6 IgG responses and individual exposure to Anopheles bites. Encouraging data has accumulated for the use of the gSG6 salivary protein as serological marker of exposure to the Anopheles genus. Incontestably, the gSG6 protein can be used to detect exposure to Anopheles bites, even in areas of low exposure[An. gambiae and An. funestus could distinguish individuals un-exposed to Anopheles bites from individuals exposed to Anopheles bites and could also distinguish high levels of exposures, independent of the anopheline Cellia species fauna. However, gSG6 was found to elicit a higher level of response than fSG6 orthologs. Overall, the analysis of IgG responses against SG6 ortholog proteins sustains the use of SG6 proteins as consistent indicators of exposure to three major malaria vectors in tropical Africa .The well-conservedSG6 protein family within the exposure. In the An. funestus densities. These initial tests provided encouraging preliminary information on the immunogenicity of the anopheline 5′nuc proteins and present the promising possibility of using the 5′nucleotidase salivary protein from An. funestus as the first species-specific antigenic marker of exposure. Complementary studies are needed to confirm the present assumption.In contrast, the presence of significantly different patterns and intensities of IgG responses against 5′nucleotidase anopheline orthologs supports the idea that each of these proteins should possess specific antigenic epitopes. Moreover, the IgG response level against the f-5′nuc protein seems to be associated with ACN: Acetonitrile; aOD: adjusted optical density; CI: Confident interval; CV: co-efficient of variation; ELISA: Enzyme-Linked ImmunoSorbent Assays; HBR: Human biting rate; MS: Mass spectrometry; NCBI: National Center for Biotechnology Information; SD: Standard deviation; SG6: Salivary gland protein 6.The authors declare that they have no competing interests.AL and RC conceived and designed the experiments. ZA and BM performed the experiments. AL, FA, RC, FC and PF analysed the data. BN, AS, BY, RF, VHV contributed reagents/materials/analysis tools. AL, FC, FA and RC wrote the paper. All authors read and approved the final manuscript.Paired-wise alignment of 5′-nucleotidase proteins from An. gambiae and An.funestus.Click here for fileComparison of sequence alignment of Culicidae protein members from the 5′ nucleotidase/Apyrase family to 5′-nucleotidase proteins from An. gambiae (gi|4582528).Click here for filePhylogram tree constructed from the alignment of the SG6 protein sequences from Anopheles species.Click here for fileStatistical analysis of variations in IgG responses per site against the anopheline salivary proteins.Click here for file


Mycobacterium leprae. In the pathogenesis of leprosy, granulomas play a key role, however, the mechanisms of the formation and maintenance of M. leprae granulomas are still not clearly understood.Leprosy is a contagious and chronic systemic granulomatous disease caused by M. leprae granulomas and the interaction between the bacilli and human host cells, we developed an in vitro model of human granulomas, which mimicked the in vivo granulomas of leprosy. Macrophages were differentiated from human monocytes, and infected with M. leprae, and then cultured with autologous human peripheral blood mononuclear cells (PBMCs).To better understand the molecular physiology of M. leprae infected macrophages were co-cultured with PBMCs. Histological examination showed M. leprae within the cytoplasmic center of the multinucleated giant cells, and these bacilli were metabolically active. Macrophages of both M1 and M2 types co-existed in the granuloma like aggregates. There was a strong relationship between the formation of granulomas and changes in the expression levels of cell surface antigens on macrophages, cytokine production and the macrophage polarization. The viability of M. leprae isolated from granulomas indicated that the formation of host cell aggregates benefited the host, but the bacilli also remained metabolically active.Robust granuloma-like aggregates were obtained only when the in vitro model of human M. leprae granulomas was established using human monocyte-derived macrophages and PBMCs. This system may be useful to unravel the mechanisms of disease progression, and subsequently develop methods to control leprosy.A simple  Mycobacterium leprae through the fine-tuning between cells, cytokines and chemokines continues to be unraveled. Similar to other mycobacterial infections, granulomatous inflammation in the skin lesion defines certain forms of leprosy [M. leprae granulomas vary with the status of the host immune response. Presumptively, granulomatous lesions can be categorized within two polar forms [Leprosy is a chronic mycobacterial infection that presents an extraordinary range of cellular immune responses in humans. Regulation of cell-mediated immunity against  leprosy ,2. The b leprosy . The orgar forms . At one M. marinum and M. tuberculosis suggested that the granulomas contribute to early bacterial growth and expanding infection [Granulomas have long been believed to benefit the host by containing and restricting the growth of mycobacteria in a localized area, to prevent the spread of the disease to other parts of the tissue or organs . Howevernfection -10.in vitro models of human mycobacterial granulomas have been studied by infection with Bacillus Calmette-Guérin (BCG) or stimulation with antigens such as purified protein derivatives or artificial beads coated with mycobacterial components [The structure, function, and evolution of granulomas have been studied using various animal models ,12, highmponents ,16. Thesmponents -19. Howein vitro model of M. leprae granulomas, which mimicked the human granulomatous skin lesion with progressive recruitment of monocytes around macrophages infected by M. leprae, and their differentiation into ECs and MGCs as well as recruitment of activated lymphocytes. This model may be useful for unravelling the mechanisms of disease progression, and find effective strategies to control the spread of bacilli.In this study, we developed an M. leprae (Thai-53 strain) was propagated in athymic BALB/c-nu/nu mice  [M. leprae[M. leprae was freshly prepared. Human cells without the bacilli were cultured at 37°C but when infected with the bacilli, the cells were cultured at 35°C to maintain the viability of M. leprae in host cells.Peripheral blood was obtained from healthy Japanese individuals with informed consent. The study was approved by the ethics committee of the National Institute of Infectious Diseases (NIID). In Japan, BCG vaccination is compulsory for children aged 0–4 years old. Macrophages were differentiated from monocytes using granulocyte-macrophage colony-stimulating factor (GM-CSF) as described previously ,21. Anim, Tokyo) . At 8–9 M. leprae. For all5 cells/well). Freshly prepared M. leprae were then added to each well. The multiplicity of infection (MOI: 50) was determined based on the assumption that macrophage were equally susceptible to infection with M. leprae[M. leprae infected macrophages at a ratio of 5:1 (PBMCs:macrophages). In some cases, macrophages were infected with M. leprae without PBMCs and in others, macrophages and PBMCs were co-cultured and macrophages alone were used as negative controls. The cells were cultured at 35°C for periods from 24 h to 10 days with medium changes every other day. To detach the cells from plates TrypLE Express (Gibco) was used, and then the cells were maintained in medium containing 10%FCS for 30 min, before processing for flow cytometric analyses. In other experiments we have also isolated T lymphocytes and monocytes were isolated using Dynabeads Untouched Human T cells and Dynabeads MyPure Monocyte kit 2 (Invitrogen), and used instead of PBMCs.Macrophages, differentiated from monocytes using GM-CSF after 4 days culture in RPMI containing 20% fetal calf serum (FCS) were transferred into 24-well tissue culture plates  . Photographs were taken with an Olympus DP50 system. Image acquisition and data processing were performed using DP controller software. In other experiments, cells were stained with May-Grünwald-Giemsa stain (MGG) (Sigma-Aldrich) or by TB Carbolfuchsin ZN stain according to the manufacturer’s instructions (BD Biosciences).Macrophages grown on a 13-mm coverslip in a 24-well plate, were infected with M. leprae was stained by auramine O (BD Biosciences). Images were obtained under a fluorescence confocal microscope. Data were processed using LSM software ZEN 2007.Cell imaging was performed using LSM5-Exciter laser scanning microscope equipped with a 568 nm laser (Carl Zeiss). Fixed cells were stained with anti-human CD163 monoclonal antibody (mAb: BioLegend) and the secondary antibody used was an Alexa Fluor 568-conjugated goat anti-mouse IgG (Invitrogen/Molecular Probes). Nuclei were counterstained with Hoechst 33342 dye (Sigma-Aldrich). M. leprae stimulation. The expression of cell surface antigens on macrophages, was analyzed using a FACSCalibur flow cytometer (BD Biosciences). Dead cells were eliminated from the analysis by staining with 7-amino actinomycin D. For the analysis of cell surface antigens, the following mAb were used: FITC-conjugated mAb against CD68 (KP) was purchased from Dako, FITC conjugated TLR4 (HTA125) and CD206 (19.2), and PE conjugated mAb against CD86 (FUN-1) was all purchased from BD Biosciences and PE conjugated mAb to CD14 (HCD14) and CD163 (RM3/1) were from BioLegend. The numbers in the insets indicate the mean fluorescent values of the cells stained with the respective mAbs.Macrophages were collected after time points of 1 and 9 days of co-culture with the PBMCs or The levels of the cytokines: Interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF)-α, IL-12p40, IL-1β and IL-10 in the culture supernatants were quantified using enzyme assay kits, OptEIA Human ELISA Set (BD Biosciences) and processed according to the manufacturer’s instructions. IL-4 and IL-13 was purchased from MABTECH AB. Cytokine levels were expressed as pg of protein/ml of protein. Real-time PCR analysis of mRNA extracted using an RNeasy Mini kit (Qiagen), was performed using SYBR Green PCR Master Mix (Applied Biosystems) with specific primers according to the manufacturer’s instructions. The instrument used for the detection of the expression of mRNA was StepOnePlus with StepOne software.M. leprae recovered from the macrophages of different groups was detected by radiorespirometry, that measures the oxidation of 14C palmitic acid to 14CO2, as described previously [M. leprae. After neutralization with 0.1 N HCl solution, an equal volume of 2 times concentrated Middlebrook 7H9 broth was added. 14C labeled palmitic acid was added to the lysates of macrophages or granulomas, followed by incubation at 33°C. After 7 days, cumulative amounts of oxidized palmitic acid released as 14CO2 by metabolically active M. leprae were measured using a Packard 1500 TRI-CARB liquid scintillation analyzer. The unpaired Student’s t-test was used to determine the statistical significance of the two data sets.The viability of eviously . BrieflyM. leprae infected macrophages in a 24-well tissue culture plate, the cells aggregated to form a multilayered granuloma-like aggregates by day 9 as shown in Figure in vitro granulomas exhibited a cellular structure similar to that in histopathological specimens of tuberculoid leprosy lesions showing T lymphocytes surrounding the differentiated, ECs and MGCs that may be involved in cytokine production for intercellular communication. , and some cells were positive for CD163 (red), a macrophage marker , CD68 (macrophage marker related to phagocytic activities), CD163 (scavenger receptor) and CD206 (mannose receptor), although the expression of major histocompatibility complex (MHC) class-II, CD86, and toll-like receptor (TLR)-4 did not change  and mannose receptor (CD206) molecules. CD163 and CD206 are the markers of M2 macrophages. Therefore, the M1 and M2 macrophages coexisted in granuloma-like aggregates. Consistent with this observation, the levels of IL-1β, IL-2, IL-12 and IFN-γ were high in the culture medium, promoting the differentiation of macrophages into both M1 and M2 subsets. The protective cell mediated immune response is regulated by the cytokine equilibrium, while the tuberculoid pole is characterized by the presence of Th1 cytokines , and lepromatous is characterized by type 2 cytokines  [M. leprae suppressed the production of IL-10. However, when macrophages were differentiated with M-CSF, rather than GM-CSF, M. leprae infection further enhanced IL-10 production. Our results indicate that the granuloma aggregates studied here, are similar to those observed in the tuberculoid form of leprosy. However, little is known about the type of cytokines that influence the formation of macrophages for containment of M. leprae in the granulomas during the pathogenesis of leprosy.Macrophages function as control switches of the immune system, providing a balance between pro- and anti-inflammatory responses by developing into subsets of M1 or M2 activated macrophages. M1 macrophages are activated by type I cytokines such as IFN-γ and TNFα, Alternatively, activated M2 macrophages are subdivided further into M2a (activated by IL-4 or IL-13), M2b (activated by immune complexes in combination with IL-1β) and M2c (activated by IL-10 or glucocorticoids) . M1 macrd IL-10) . BecauseM. leprae in macrophages at different time points. At day 9, a number of granuloma-like aggregates were observed in co-cultures of PBMCs with macrophages infected with M. leprae. However in macrophages infected with M. leprae without the PBMCs, granuloma-like aggregates were not observed. There were no significant differences in the viability of M. leprae in macrophage of different groups on day 1, but on day 9, the viability of M. leprae in the group that formed granuloma-like aggregates was slightly lower, although not significantly, than that of M. leprae in infected macrophages without PBMCs. Evidently, granuloma-like aggregates appear to benefit the host but the bacilli remained metabolically active. The mechanism of this phenomenon needs further in-depth analysis.We also investigated the viability of in vitro M. leprae granulomas using human monocyte derived macrophages and PBMCs. Using this model, we obtained some basic information about the characteristics of in vitro granulomas. In addition, the viability of M. leprae in granuloma-like aggregates remained unaltered during the culture period. Effective strategies to allow the bacilli to succumb to the formation of granuloma may assist in the primary control of the infection.In summary, we have developed for the first time a method to obtain DCs: Dendritic cells; PBMCs: Peripheral blood mononuclear cells; ECs: Epitheloid cells; MGCs: Multinucleated giant cells; BCG: Bacillus Calmette- Guérin.The authors declare that they have no competing interests.HW, YM participated in the design of the study and carried out the cell culture experiments, YF carried out the confocal microscopic examination, and radio-respirometric assay. HW, YM, and MM were involved in the preparation of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/279/prepubMeasurement of IL-10 secreted into the culture medium by ELISA. Measurement of IL-10 secreted in the culture medium from different groups of cells at days 2, 6 and 9 is shown. Two types of macrophages were used to analyze the data. (A) Macrophages differentiated using GM-CSF, and (B) macrophages differentiated from monocytes using M-CSF. Representative data from two individual experiments of a single donor are shown. Unpaired student’s t test was performed, *p < 0.0001, **p < 0.001, ***p < 0.05.Click here for file


Hansenula fabianii J640. The promoter and terminator regions of the gene encoding glucoamylase from H. fabianii J640 were used for a new expression vector, pHFGE-1. The performance of pHFGE-1 was compared with that of the widely used pG-1 transformant vector. H. fabianii J640 (HF-TAMY) cells were transformed with pHFGE-1, and Saccharomyces cerevisiae YPH-499 (SC-TAMY) cells were transformed with pG-1, both of which carried the Taka-amylase. Expression of Taka-amylase by HF-TAMY showed higher than that by SC-TAMY. By using this new system, we bred the new wastewater treatment yeast that shows α-amylase activity. This yeast appears to grow well under experimental wastewater conditions, and is effective in treating model wastewater containing soluble and insoluble starch.We previously developed a host vector system for the wastewater treatment yeast  Many food factories use wastewater treatment systems that use yeasts . HoweverCryptococcus sp. S-2 . Then, we constructed a new expression vector, pHFGE-1 , which uses pHFura3, and the promoter and terminator regions of the gene encoding glucoamylase from H. fabianii J640. We inserted the genes encoding α-amylase and xylanase from Cryptococcus sp. S-2 between the promoter and terminator of pHFGE-1. When the pHFGE-1 with one or the other of these foreign genes were transformed into H. fabianii J640 u-1, the transformants  showed α-amylase and xylanase activities respectively. This showed that pHFGE-1 can derive the expression of foreign genes in H. fabianii J640 cells.We purified the glucoamylase of In this paper, we investigated the ability of these transformed yeasts, to treat wastewater, and developed a PCR method for monitoring the presence of the foreign gene.H. fabianii J640 and Cryptococcus sp. S-2 were obtained from the National Research Institute of Brewing culture collection, Japan. A uracil auxotrophic mutant of H. fabianii J640, named H. fabianii J640 u-1, lacking orotidine-5'-phosphate decarboxylase, was used as a host strain for new expression vector pHFGE-1. S. cerevisiae YPH-499 (MATα ura3 lus2 ade2 trp1 his3 leu2) was used as the host for transformation vector pG-1  without amino acids was used to select the yeast transformants. YPM medium was prepared by replacing the glucose of YPD with maltose. The medium used to investigate expression induction, contained 1% yeast extract, 1% casamino acid, and 2% glucose or maltose.Yeast cells were grown on YM medium  and YPD medium . Luria-Bertani medium containing ampicillin (100 μg/ml) was used to cultivate BamHI site between the promoter and terminator from H. fabianii J640 glucoamylase DNA. The host cell of this vector is a uracil auxotrophic mutant designated as H. fabianii J640 u-1, and it could be transformed by a non-homologous and frequently multicopy integration into the host genomic DNA.The expression vector pHFGE-1  Figure  was usedTransformations were carried out by electroporation as described by . ElectroXylanase activity was assayed by measuring the amount of reducing sugar liberated from xylan . One uni5-azide-65-deoxy-β- maltopentaoside under the condition described above.α-Amylase activity was measured with an α-amylase kit (Kikkoman). One unit of α-amylase activity was defined as the amount of enzyme which forms 1 μmol of 2-choloro-4-nitrophenol from 2-choloro-4-nitrophenyl 6Model wastewater containing soluble starch was made with 1% refined starch (Merck) and 0.25% yeast extract, pH 6.0. The starch was solubilized by autoclaving. Model wastewater containing insoluble starch was made with 0.25% yeast extract, pH6.0, autoclaved and cooled to approximately 55°C. The same amount of starch was sterilized in 70% ethanol. The suspension was centrifuged and decanted. The starch pellet was then added to the autoclaved yeast extract solution.6 cells/ml was inoculated to the model wastewater in an Erlenmeyer flask. Cultures were incubated at 30°C with shaking at 105 rpm and samples were periodically harvested.Yeast cells were incubated at 30°C for 2 days on YM medium. Then 5 × 10The model wastewater containing soluble starch was centrifuged at 3000 rpm for 10 min, and chemical oxygen demand (COD) of the supernatant was measured. The decrease in COD of the model wastewater containing soluble starch was used to express the capacity of the yeast to treat the wastewater.It was not possible to measure COD of the model wastewater containing insoluble starch because of the difficulty in separating the cells and insoluble starch. In this case, degradation of the starch was measured with the iodo-starch reaction  as folloThe transformants were cultured in 10 ml YM medium and harvested by centrifugation. DNA was extracted with an Easy-DNA kit (Invitrogen) and used for the PCR template. Unique PCR primers were designed, and the position of the PCR product is shown in Figure 6-101 cells/ml). One ml of each dilution was harvested and DNA was extracted with the EASY-DNA kit and used as a PCR template.To determine the sensitivity of the PCR, cells were cultured in YM medium, and the cell density was measured. Then a dilution series was made  cells were transformed with pHFGE-1, and S. cerevisiae YPH-499 (SC-TAMY) cells were transformed with pG-1, both of which carried the Taka-amylase gene. The cells were then cultured on YPD and YPM media. Growth on YPD medium was the same for the two cultures ), only the transformant showed a PCR product . The expression of the foreign gene that was integrated in the transformant was induced by maltose and repressed by glucose. However, the growth rates of the transformants carrying pHFGE-1 and the widely used pG-1 were the same and both transformants strongly expressed the foreign gene, even in medium containing glucose, which was expected to repress expression of the foreign gene. Our host vector system strongly expresses the foreign gene. Because wastewater contains various components, the strong expression of the new strain is an advantage. The HF-AAMY cells were effective in treating the model wastewater.We developed a host vector system for the wastewater treatment yeast, Because HF-AAMY cells are genetically modified, a sensitive method for monitoring the cells in the environment is needed. Our PCR was shown to satisfy this requirement.Hansenula polymorpha (H. polymorpha is rapidly becoming the system of choice for heterologous gene expression in yeast. Several production processes for recombinant pharmaceuticals and industrial enzymes have been developed based on gene expression in this strain. Another methylotrophic yeast, Hansenula ofunaensis, has also been evaluated for a transformation system (A host vector system was also developed for the methylotrophic yeast lymorpha . As in tn system  but deveHansenula anomala, another wastewater treatment yeast, was developed in the 1990s (A transformation system using he 1990s . HoweverOur next goal is to use our transformant to treat real wastewater from the food industry. In the future, when genetically engineered yeast is proven to be effective for the treatment of wastewater, a major task will be to prove to the public that the methodology is safe.The authors declare that they have no competing interests.


Sini decoction (SND) is a well-known formula of Traditional Chinese Medicine (TCM) and is considered as efficient agents against DOX-induced cardiomyopathy. However, its action mechanisms are not well known due to its complex components.Doxorubicin (DOX) is one of the most potent antitumor agents available; however, its clinical use is limited because of the risk of severe cardiotoxicity. Though numerous studies have ascribed DOX cardiomyopathy to specific cellular pathways, the precise mechanism remains obscure. SND, which showed that SND administration could provide satisfactory effect on DOX-induced cardiomyopathy through partially regulating the perturbed metabolic pathways.A tissue-targeted metabonomic method using gas chromatography–mass spectrometry was developed to characterize the metabolic profile of DOX-induced cardiomyopathy in mice. With Elastic Net for classification and selection of biomarkers, twenty-four metabolites corresponding to DOX-induced cardiomyopathy were screened out, primarily involving glycolysis, lipid metabolism, citrate cycle, and some amino acids metabolism. With these altered metabolic pathways as possible drug targets, we systematically analyzed the protective effect of TCM The results of the present study not only gave rise to a systematic view of the development of DOX-induced cardiomyopathy but also provided the theoretical basis to prevent or modify expected damage. Doxorubicin (DOX) is a powerful anticancer antibiotic Sini decoction (SND), which is officially recorded in the Chinese Pharmacopoeia 2010 edition and has been used to prevent or treat cardiovascular disease as well as DOX-induced cardiomyopathy for many years Acontium carmichaeli, Glycyrrhiza uralensis and Zingiber officinale. As there are complex components in SND, which can hit multiple targets and exert an overall therapeutic effect, it is hard to understand its action mechanisms completely.Medicinal plants play a key role in human health care nowadays. It is increasingly being understood that the Traditional Chinese Medicine (TCM) does have a remedy for acute or chronic disorders. Metabonomics, the systems biology approach of small molecules, is a rapidly advancing field that aims to monitor as many metabolites with low molecular weight as possible rather than focus on individual metabolite in a single cell, biofluids, and tissue extracts SND and its mechanisms were also investigated by using a metabonomic approach for the first time.Though a lot of urinary biomarkers have been successfully identified from the systemic toxicity of doxorubicin, the tissue-specific biomarkers in myocardium still need further study to provide a new insight into the pathological processes of doxorubicin-induced cardiomyopathy. Therefore, a gas chromatography–mass spectrometry (GC-MS) method based on a metabonomic strategy was developed. In addition, the reverse effects of TCM SND significantly attenuated DOX-induced increase in the above enzymes in serum, indicative of reduced myocardial necrosis (p<0.05).Serum levels of creatine kinases (CK), creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were significantly elevated 72 h after DOX injection compared with the levels in the control rats , the data was simplified and 170 ion peaks were obtained. The unsupervised Principal Component Analysis (PCA) was first applied to explore correlations between control and DOX groups, and a tendency in the score plot to separate the two classes was detected (R2 = 0.50) (p<0.05). Finally, it was found that sixteen of 24 metabolites, including L-alanine, phosphate, glycine, succinate, malate, proline, threonic acid, glutamine, phenylalanine, dihydroxyacetonephosphate (DHAP), glycerol-3-phosphate (G-3-P), fructose, glucose, stearic acid, myo-inositol and cholesterol, were evaluated in the DOX group, while the other eight metabolites including lactate, β-hydroxybutyric acid, L-valine, isoleucine, threonine, citrate, linoleic acid, arachidonic acid, were reduced in the DOX group.  of myocardial extracts are shown in etected R2 = 0.50 etected R2 = 0.50 X group. .SND for protection of DOX-induced cardiomyopathy have been shown in Section “Serum biochemical assay”. As the 24 potential biomarkers have been found, it is reasonable to take them as the monitoring indexes for further investigating the protective mechanisms of SND to DOX-induced cardiomyopathy. Therefore, the levels of these 24 biomarkers were introduced as variables to PCA, performed on three groups. As shown in SND group was closer to the control group, which might suggest SND can reverse the pathological process of DOX-induced cardiomyopathy. To further evaluate the reversed condition of the potential biomarkers by pre-administration with SND, one-way ANOVA with Tukey post hoc test was performed by SPSS software. The ratios of relative peak area of the 24 metabolites to that of ribitol are shown in SND group. The other fourteen metabolites were also reversed at different degrees.The effective results of SND could play its role in DOX-induced cardiomyopathy by intervening in some metabolic pathways, such as glycolysis, lipid metabolism, citrate cycle, and some amino acids metabolism, which might be mapped by restoring alterations in some biomarkers found in this research.The present study demonstrates, for the first time, a GC-MS-based metabonomic analysis of DOX-induced cardiomyopathy in mice, identifying 24 potential biomarkers relevant to the disturbance of myocardial energy metabolism primarily involving glycolysis, lipid metabolism, citrate cycle, and some amino acids metabolism. Furthermore, it was confirmed that Chinese Medicine et al.Among these metabolites, lactate, fructose, glucose, DHAP and G-3-P are correlated with glycolysis, indicating the modulation of the glycolytic pathway. A reduced in lactate level was observed in DOX-induced group. It is conceivable that the reduction should be accompanied with the regulation of enzymes in the heart. The lactate dehydrogenase B (LDHB), a subtype of lactate dehydrogenase mainly existing in myocardium, was recently found to be up-regulated in the DOX treated cardiomyocytes et al. supported that the activities of SDH and MDH indeed decreased in DOX-treated rats The citrate cycle is central to aerobic metabolism, facilitating adequate throughout of substrates derived from carbohydrates, fatty acids or certain amino acids Lipid metabolism has a key role in DOX-induced cardiomyopathy, which has been demonstrated by numerous studies The low levels of valine and isoleucine, two branched-chain amino acids (BCAAs), were observed in DOX group. BCAAs may be an important alternative energy substrate for the heart in myocardial ischemia SND could up-regulate B-cell lymphoma-extra large (Bcl-xl) protein and down-regulate BH3 interacting domain death agonist protein in DOX-induced cardiomyocytes SND. In this study, the down-regulation of succinate, malate, and stearic acid and up-regulation of lactate and citrate were observed in DOX plus SND group compared with DOX group, which implied that SND might functionally intervene in glycolysis, lipid metabolism and citrate cycle. In fact, SND administration permitted the mean levels of each potential biomarker to reverse at different degrees. These results combined with the serum biomedical assay suggested that SND has unique characteristics for the protective effect on DOX-induced cardiomyopathy. The potential biomarkers revealed metabolic pathways  might be involved in the protective mechanism of SND. Based on these discussions, the schematic overview of the potential biomarkers changing for DOX-induced cardiomyopathy in cardiac muscle and SND modulation is shown in SND.It was reported that SND. Taking the potential biomarkers found in the study as screening indexes, it revealed that SND could restore the unbalanced glycolysis, lipid metabolism, citrate cycle, and amino acids metabolism. Therefore, it is suggested that TCM SND was a promising potential cardioprotection agent against DOX-induced toxicity.In conclusion, a metabonomic study of the biochemical profiles of DOX-induced cardiomyopathy was performed using GC-MS and multivariate analysis. Such a study allowed the holistic evaluation of changes in the level of metabolites. With Elastic Net for classification and selection of biomarkers, twenty-four metabolites primarily involved in glycolysis, citrate cycle, lipid metabolism and amino acids metabolism, were screened out and considered as potential biomarkers corresponding to DOX-induced cardiomyopathy. Combined with the previous studies, the metabolites' change shed new light on DOX cardiomyopathy, which would be used to understand the development of the disease and assess drug efficacy as well as and mode-of-action. The GC-MS-based metabonomic method has been successfully applied to evaluate the protective effect of TCM All animal experiments were approved by the Administrative Committee of Experimental Animal Care and Use of Second Military Medical University (SCXK(Hu)2007-0005), and conformed to the National Institute of Health guidelines on the ethical use of animals.N-methyl-N-(trimethylsilyl)- trifluoracetamide (MSTFA), pyridine, trimethyl-chlorosilane (TMCS), n-heptane, acetone, β-hydroxybutyric acid and citrate were purchased from Sigma-Aldrich . Methanol and acetonitrile are chromatography pure . The following compounds were obtained from Shanghai Jingchun Reagent Co.: ribitol, lactate, L-alanine, L-valine, phosphate, isoleucine, glycine, Sodium succinate, threonine, malate, L-proline, L-threonic acid hemicalcium salt, L-glutamine, phenylalanine, fructose, glucose, myo-inositol, linoleic acid, stearic acid, arachidonic acid and cholesterol.The assay kits for creatine kinases (CK), creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were purchased from Shanghai Fosun Long March Medical Science Co. Methoxylamine hydrochloride, Acontium carmichaeli , Glycyrrhiza uralensis  and Zingiber officinale  were purchased from Shanghai Dekang Medicine Corp.  were authenticated by Lianna Sun .SND recorded in Chinese Pharmacopoeia 2010 edition, it was prepared using the following procedure. The crude drugs of A. carmichaeli 60 g, G. uralensis 60 g and Z. officinale 40 g were immersed in 1.6 liter water for 1 h and then decocted to boil for 2 h. The decoction was filtered through four layers of gauze. Next, the dregs were boiled once again for 1 h with 1.2 liters of water and the decoction was filtrated out with the above method. Afterward, the successive decoctions were merged and condensed by a rotatory evaporator under reduced pressure. Finally, the extraction solution was made to a concentration of 1.0 g/mL . According to our previous published paper SND were identified.According to the original composition of ad libitum throughout the experimental period. The animals were randomly divided into three groups (n = 8). Control group: 200 µL of normal saline were injected i.p. DOX group: animals of the DOX group received a single dose of DOX . DOX plus SND group: the mice were treated with SND 10 g/kg/BW o.p. for 7 consecutive days as follows: 4 days before, on the same day, and 2 days after administration of a single dose of DOX . The administration dose in the current study is in accordance with clinical use. Seventy-two hours after DOX administration, blood samples were collected for biomedical measurement including CK, CK-MB and LDH and hearts were rapidly excised and frozen in liquid nitrogen for metabonomic analysis.All animal experiments were performed at the Centre of Laboratory Animals of the Second Military Medical University  in accordance with the relevant national legislation and local guidelines. ICR mice, 25∼30 g weight, were purchased from the Shanghai Laboratory Animal Co. Mice were housed with temperature range from 20°C to 25°C and humidity range from 50% to 60%. The mice were fed with commercial aseptic food and tap water 2 stream at room temperature. The derivatization was performed using methoxyamine pyridine  at 70°C for 1 h, followed by MSTFA (75 µL) with 1% TMCS as catalyst at room temperature for 1 h. n-Heptane (200 µL) was added to dilute the solution after the derivatization and the supernatant was used for GC-MS analysis.Myocardial tissue (∼50 mg) was homogenized in 200 µL saline and then centrifuged at 13,000×g for 15 min at 4°C. 100-µL aliquot of the supernatant was transferred into a centrifuge tube and spiked with the internal standard (5 µL of 1 mg/ml of ribitol), followed by adding 400 µL ice cold methanol ∶ acetonitrile ∶ acetone (1∶1∶1) into the tube. After vigorous shaking for 30 s and incubation on ice for 10 min, the mixture was centrifuged at 14,000×g for 15 min to precipitate the protein. The supernatant (300 µL) was transferred into a GC vial and evaporated to dryness under NThe derivatized samples for GC-MS were analyzed on a Thermo-Finnigan Trace DSQ fasting scanning single-quadrupole MS (Thermo Electron Corporation) operated at unit mass resolving power. A 1.0 µL of sample solution was injected with splitless mode to TB-5MS column (30 m×250 µm×0.25 µm) with helium as the carrier gas at a flow of 1.0 mL/min. The injector temperature was set at 260°C. The temperature of the ion source was adjusted to 200°C and that of quadrupole was set at 150°C. GC-MS was operated using electron impact ionization with a 60–600 atomic mass unit (amu) scan range. The initial temperature of column was kept at 70°C for 4 min. Then the temperature was ramped at 4°C /min to 220°C. Subsequently, the temperature was increased to 310°C by 8°C /min and was held for 10 min. Identification of the interested peaks was performed by searching the NIST database installed in the Thermo-Finnigan Trace DSQ GC-MS system and comparing with the peaks of commercial standards.In order to validate the stability of the GC-MS system, an equal volume of each sample was pooled together to generate a pooled quality control (QC) sample m/z 216.9, it is the most abundance fragment ion for the silylation derivative of ribitol).Data in instrument-specific format were converted to CDF format files. The program XCMS was used for nonlinear alignment of the data in the time domain and automatic integration and extraction of the peak intensities It should be noted that GC-MS data inherently contain apparent variability and complexity such as multiple fragment ions from a single compound. Directly concatenating the matrices of processed-MS data is suboptimal as this may result in a matrix with an unfavorable samples-to-variables ratio p<0.05.Statistically significant differences in mean values were tested by one-way ANOVA and the Tukey post hoc test for comparisons of multiple groups. The differences were considered significant when SND groups based on myocardial metabolites. The Elastic Net using the library “glmnet” in the R environment (http://www.r-project.org) was used for classification and selection of significant ion peaks between control and DOX groups. The significant peak changes between samples were confirmed by manual quantification by calculating the area under the peak from raw chromatograms.Prior to multivariate analysis, the data list was log-transformed and normalized. The principal component analysis (PCA) using SIMCA-P  was used to uncover unknown trends in the control, DOX and DOX plus Table S1The relative levels and one-way ANOVA results of the potential biomarkers in the myocardial tissues of control, DOX and DOX plus SND group.(DOC)Click here for additional data file.


To evaluate if human papillomavirus (HPV) self-sampling (Self-HPV) using a dry vaginal swab is a valid alternative for HPV testing.Women attending colposcopy clinic were recruited to collect two consecutive Self-HPV samples: a Self-HPV using a dry swab (S-DRY) and a Self-HPV using a standard wet transport medium (S-WET). These samples were analyzed for HPV using real time PCR (Roche Cobas). Participants were randomized to determine the order of the tests. Questionnaires assessing preferences and acceptability for both tests were conducted. Subsequently, women were invited for colposcopic examination; a physician collected a cervical sample (physician-sampling) with a broom-type device and placed it into a liquid-based cytology medium. Specimens were then processed for the production of cytology slides and a Hybrid Capture HPV DNA test (Qiagen) was performed from the residual liquid. Biopsies were performed if indicated. Unweighted kappa statistics (к) and McNemar tests were used to measure the agreement among the sampling methods.A total of 120 women were randomized. Overall HPV prevalence was 68.7%  59.3–77.2) by S-WET, 54.4% (95% CI 44.8–63.9) by S-DRY and 53.8% (95% CI 43.8–63.7) by HC. Among paired samples (S-WET and S-DRY), the overall agreement was good  and the κ was substantial . The proportion of positive type-specific HPV agreement was also good . No differences in sensitivity for cervical intraepithelial neoplasia grade one (CIN1) or worse between the two Self-HPV tests were observed. Women reported the two Self-HPV tests as highly acceptable.Self-HPV using dry swab transfer does not appear to compromise specimen integrity. Further study in a large screening population is needed.NCT01316120ClinicalTrials.gov:  Cervical cancer incidence and mortality have decreased considerably since the introduction of cervical cancer screening programs in Western countries. However, despite these advances in secondary prevention, there are 500,000 new cases every year worldwide, mostly 85%) in developing countries -3. Cervi5% in devAcceptability studies for Self-HPV indicate that the method is generally well accepted by women, but revealed that some of women have doubts about the validity of the method. One of these was the concern about manipulating the test tube and spilling out the transport medium during the sampling procedure, which some patients interpreted as incorrect and feared that it might affect the test result ,13. ThisThis study was conducted by the Geneva University Hospitals, Switzerland. The Ethics Committee of the Geneva University Hospitals, Switzerland, approved the study . A signed informed consent form (ICF) was required for enrollment of participants in the study. This trial was registered at ClinicalTrials.gov (Identifier: NCT01316120).A total of 120 women were prospectively enrolled from our colposcopy clinic between November 2010 and August 2011. We randomized the sequence of the two HPV tests to avoid any potential biases that may advantage the first test. We included women aged 20 years or older, who understood the study procedures and accepted to participate by signing the ICF. Exclusion criteria were pregnancy and previous conization or hysterectomy.The participants were randomized and received oral instructions by a physician or a research nurse about how to perform the Self-HPV, a self-collected vaginal sample. In brief, they were instructed to wash their hands before performing the procedure, to insert the swab into the vagina and to rotate it three times in both directions. The women were handed two self-sampling kits ) and were directed to a private, well-lit room to perform both samplings. Subsequently the participants were asked to complete a questionnaire on demographic characteristics, knowledge about HPV and preference between the two Self-HPV methods. The questionnaire used a 4-point scale to measure the degree of acceptability, physical discomfort and pain felt using the Self-HPV as well as the preference between the two Self-HPV methods. Participants then underwent a colposcopic examination. During this procedure, a cervical sample for liquid-based cytology and Hybrid Capture (HC) HPV test was obtained using a broom type cervical brush and rinsing it in Preservcyt™ buffer solution . A biopsy for histological analysis was performed if necessary.Self samples were stored at room temperature and the time between sample collection and analysis ranged between 5 to 15 days. HPV test according to the manufacturer’s recommendations. This assay is an automated DNA extraction, PCR amplification and real-time detection of 14 High Risk HPV (HR-HPV) genotypes. It uses the beta-globin gene as an internal extraction and amplification control. PCR amplification and detection are performed in a single tube, where probes with four different reporter dyes track the different targets in the multiplex reaction: (i) HPV 16, and 18 individually, (ii) 12 HR-HPV types  as a group, and (iii) beta-globin.Material from dry swabs (S-Dry samples) was placed into 1 ml of sterile phosphate-buffered saline (PBS)and the tubes were vortexed for 3×15 sec. Then, 0.5 ml of each sample was used for nucleic acid extraction and the rest was frozen at −20°C for storage. Tubes containing S-Wet samples (1 ml) were also vortexed for 3×15 sec and then, 0.5 ml of each sample was used for nucleic acid extraction and the rest of sample was frozen at −20°C for storage. HPV extraction, detection and genotyping were carried out from S-Dry and S-Wet specimens using the Cobas 4800 Postcytology vials processed on the ThinPrep 2000 System  were used. At least 4 mL of remaining PreservCyt™ solution was used for the Hybrid Capture HPV DNA test. Samples were processed in the sample conversion kit and tested with HC2 according to the manufacturer's protocol with probe B for HR genotypes . Sample reactivity was measured in relative light units (RLU). A specimen was considered positive for HR HPV DNA if the ratio of the specimen RLU to the mean RLU of triplicates of a positive control at 1 pg per ml was >3.00. Samples with ratios between 1.00 and 3.00 were retested twice and were considered positive if 2 out of 3 results had a ratio >1.00.Both methods used in this study,  are FDA approved diagnostic methods. Because HC is considered as the reference standard to analyze physician-sampled specimen, as any methods in consideration to replace it should be as accurate as this method of screening.The sample size necessary to validate a difference in test performance of 10% or more was calculated assuming a HPV prevalence of at least 40%. The proportion of positive agreement (PPA) between paired S-WET and S-DRY samples was calculated by dividing the number of samples testing positive for HPV in both tests by the number of samples testing positive in either S-WET or S-DRY. Cohens Kappa was calculated to measure the inter-test agreement between the self-sampling methods in terms of HPV risk categories. Positive agreement between S-WET and S-DRY was calculated as described by Wolfrum et al. .One hundred twenty women were included in the study, of whom we excluded four for not having paired samples, three for inconclusive HPV test results  and one who refused further participation after performing Self-HPV. For the study analysis we included 112 women with 224 paired samples and completed questionnaires. The median age of participants was 31 years (range 21–63 years).The HPV prevalence was 68.7%  59.3–77.2) detected by S-WET, 54.4% (95% CI 44.8–63.9) by S-DRY and 53.8% (95% CI 43.8–63.7) by HC. Mono-infections with HPV 16 or 18 were identified in 19.4% of participants, and combined infections with HPV 16 or 18 and other high-risk types were identified in 36.3%. Infection with one or more HPV types other than HPV 16 or 18 was observed in 44.1% of participants. HPV 16 was detected in 49.3% of cases and HPV 18 in 9%.The overall test agreement between S-WET and S-DRY was 85.7% (95% CI 77.8–91.6), with a 79.2% positive agreement. Cohen’s kappa for inter-test agreement was 0.70 (95% CI 0.53-0.88). Positive agreement for type specific HPV was 77.3% (95% CI 68.2–84.9). The inter-test agreement was good between S-WET and S-DRY for type-specific detection of HPV 16 and non-16/18 HPV types as well as for all HPV positive cases. The inter-test agreements between HC and S-DRY and between HC and S-WET were inferior to the inter-test agreement of S-WET and S-DRY . No difference in sensitivity or specificity was found between S-WET and S-DRY.In the case of CIN 1 or higher lesions, the stratified overall test agreement was 88.6% (κ=0.70). S-DRY detected HPV in 73.9% of cases of CIN2/3, while S-WET was positive in 91.3% and HC was positive in 69.6%. The agreement between S-WET and S-DRY was highest in CIN1. The performance of both self-tests was comparable to HC samples taken by a physician.We did not observe any statistically significant difference between S-DRY and HC or between S-WET and HC Table .One hundred and twelve questionnaires were completed, and no difference in acceptability between the two Self-HPV tests was observed. Most women (96.4%) were favorable to the idea of performing self-sampling at home, while three women (2.7%) were opposed to this idea and one did not answer the question. Fifty women experienced Self-HPV as slightly to severely painful or embarrassing, but no preference could be highlighted between the two Self-HPV tests. Some of the women  evaluated one of the self-tests as more complex than the other, while the majority did not report any difference in complexity. Twenty-seven women had higher confidence in S-WET, while two had higher confidence in S-DRY, but the large majority (83/112) thought both self-tests were equally reliable.In recent years, we have learned that, with appropriate instruction, self-sampling of HPV specimens yield similar results compared with those collected by health care professionals. The high percentage of agreement for high-risk HPV between these two approaches suggests that Self-HPV is a promising alternative to physician-sampling for HPV testing and cervical cancer screening . BelinsoRecent studies reported that Self-HPV testing is more sensitive than the Pap test in detecting CIN2+ in Chineese and Mexican women of low socioeconomic status ,20. FurtThe possibility of using Self-HPV stored at ambient temperature without transport media would clearly enhance and simplify the utility of Self-HPV. This could reduce the costs of the methods, which might be attractive for low-resource countries. Moreover, it may reduce women’s concerns that the test quality is reduced by accidentally spilling out some of the transport medium ,13. We fIn this study we used different swabs for S-WET and S-DRY. The S-WET swab was a flocked swab that consists of fine and short filaments fixed at the top of the stick while the swab used the S-DRY swab was a standard Dacron swab consisting of a long filament enrolled around a stick. Krech et al. compared flocked and rayon swabs for their sensitivity to detect HPV infection and noticed that the sensitivity of flocked swabs was higher . They exA shortcoming of our study is the use of different HPV DNA detection assays for self-collected vaginal specimens and physician-collected endocervical specimens. The number of variables that differ in addition to wet vs. dry transport weakens our conclusion about the feasibility of dry transportation. Other weaknesses of our study are the small sample size and the fact that our population was recruited in a colposcopic clinic having a high rate of HPV prevalence. Even though the HPV positivity in S-WET compared with S-DRY is not statistically significant, it seems to point in the direction of S-DRY being slightly less sensitive than S-WET. Additional work is needed to conduct a study in a real context of screening with a larger sample size to determine if S-DRY and S-WET have equal sensitivity.Our study shows that Self-HPV swabs can be successfully transported in a dry state at ambient temperature without greatly altering specimen integrity. Self-sampling for HPV testing using S-DRY could be an alternative to other self-sampling methods that require “wet” transport media. Advantages of the dry method include a simplification of the method , as well as its ease of use and lower cost may be of advantage for women with restricted access to health care delivery. However, further research is needed to confirm the equality of both methods in a large screening population.ASC-US: Atypical squamous cells of undetermined significance; AUC: Area under the curve; CIN 1: Cervical intraepithelial neoplasia grade 1; CIN 2/3: Cervical intraepithelial neoplasia grade 2 or 3; HC: Hybrid Capture; HPV: Human Papillomavirus; HSIL: High-grade squamous intraepithelial lesion; ICF: Informed consent form; LSIL: Low-grade squamous intraepithelial lesion; NPV: Negative predictive value; PBS: Phosphate-buffered saline; physician-sampling: Physician collected cervical sample for HPV testing; PPA: Proportion of positive agreement; PPV: Positive predictive value; ROC curve: Receiver Operating Characteristic curves; S-DRY: Self-HPV using a dry swab; S-WET: Self-HPV using a standard wet transport medium; Self-HPV: Self-sampling for HPV testing using a vaginal swab.The authors declare that they have no competing interests.IE conducted the study and drafted the manuscript. VP designed the study. IN participated in coordinating the study. PAM and GA were responsible for the molecular biological analysis. JCP co-designed the study. MB directed the statistical analyses. SU conducted statistical analyses and contributed to the manuscript. PP co-designed the study and led the research project. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/353/prepub